Assisted reproductive technology (ART) includes such techniques as in vitro fertilization (IVF), artificial insemination (AI), intracytoplasmic sperm injection (ICSI) (other techniques using enucleated cells) and multiple ovulation and embryo transfer (MOET) (as well as other embryo transfer techniques) and is used across the animal kingdom. ART methods are generally expensive, time consuming and marginally successful given the inherent fragility of gametes when outside of their natural environments. Furthermore, the use of ART within the animal breeding industry in a commercially feasible manner is additionally challenging due to the limited availability of genetically desirable gametes (sperm or oocytes). One way to lower the cost of ART and to improve its commercial feasibility is to increase the efficiency of the involved processes by improving the viability and overall quality of gametes used in ART. Although there has been a growing interest in this field over the course of the last decade or so, there still remains a strong need to increase the overall quality of gametes for use in ART, especially when breeding focuses on pre-natal gender selection, including improving gametes viability, motility and fertility, as well as other longevity characteristics.
For example, in conventional AI, one problem limiting its commercial application in certain species is the need to use extremely high number of sperm per AI dose to ensure successful fertilization currently. In swine in particular, the need for improved sperm quality is especially strong since the typical dose of boar sperm required for successful fertilization using conventional artificial insemination techniques, such as intra-cervical insemination, is currently 1×109 sperm to 3×109 sperm.
Processing gametes such as flushed oocytes or sperm, both conventional and sex sorted, before their use in ART may add a tremendous amount of stress on the gamete cell(s) and often negatively impacts their cellular integrity and membrane structure, which in turn may be reflected in decreased viability, motility and fertility. An example of processing gametes prior to their use in ART is the sorting of sperm based on sex (known as “gender enrichment” or “sex sorting”), which is a now commonly used procedure to minimize wasted births of the wrong sex for selective breeding in the livestock industry. In some species, however, it is still cost prohibitive and can represent a financial risk to those with smaller breeding herds. Sex sorting includes processes that physically separate X and Y bearing sperm from each other into separate subpopulations, as well as processes in which sperm bearing the undesired sex chromosome in a sperm sample are selectively killed, compromised, disabled, rendered immotile, or otherwise rendered infertile by, for example, laser ablation/photo damage techniques to render a gender enriched population of sperm.
The sex sorting process severely stresses and damages the cells and produces a low percentage of useful sperm, which although capable of fertilizing matured oocytes, may have reduced viability, motility and fertility compared to unprocessed cells. Typically, sex sorting involves many harsh steps including but not limited to: the initial collection and handling of sperm ejaculate, which naturally starts to deteriorate rapidly upon collection; the staining of sperm, which involves binding of an excitable dye to the DNA or a harmful membrane selection procedure; the physical sorting of the sperm using high energy fluorescence that physically energizes the dye that is bound to the DNA, forced orientation through a narrow orifice, and application of an electrical charge to the cell; the physical collection of the cells into a receiving container, which often shocks the fragile cell upon contact; the osmotic stresses associated with dilution of the sperm droplet in collection media; and the storage of the sorted sperm usually by freezing, which is well known to raise havoc with the cell's membrane systems. Each step places the processed sperm under abnormal stress that diminishes the overall motility, viability and/or fertility of the sperm. The result can lead to less efficient samples for use in ART, such as IVF and AI, and other types of subsequent or further processing.
Even non-sorted processed sperm exhibits significant losses in fertility, viability and motility when being collected, handled and transported without freezing, and noticeably experiences significant stress when mixed with cryoprotectant and frozen and thawed. Many in the field have tried to improve methods for the use on unsorted, conventional semen to minimize loss in the handling processes associated with in vitro handling, preservation and use of semen samples.
Regardless of the processing, sperm lose their potential to fertilize when exposed to: elevated temperatures, abnormal buffers, stains, altered pH systems, physical pressurized orientation as when forced through a nozzle or when oscillated to form drops in a flow cytometer, radiation used to illuminate the DNA binding dye, physical stressors associated with separation and collection techniques, cryoprotectants, freezing, thawing and micromanipulation by the handler.
There remains a continuing need to improve current methods of ART to reduce the cost and to make the procedures more dependable, efficient, fast and efficacious and commercially feasible to those on a restricted budget, especially for smaller breeders for whom sex-selection breeding may be a high risk and expensive option. Accordingly, there is a significant need for improved sperm processing techniques.